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Position: Discussion forums > Bioscience biotechniques discussion forums > Immunology forum and cytometry forum
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Western Blot
Posted by: Guilherme (IP Hidden, Unregistered user, )
Date: April 14, 2005 03:46PM
Hello everyone,
I am doing western blot using a recombinant protein. I transfer the protein to nitrocellulose membrane and confirmed by Ponceau-S staining. The secondary antibody is HRP mouse anti-human IgG1 (1:5000).I donīt know why my secondary antibody, recognize all the protein. I am thinking to use the HRP goat anti-human IgG1, because when I use goat anti-human IgG my western blot works. What do you think about this? Thanks in advance, Guilherme
Immunoprecipitation Troubleshooting
Posted by: biochemistvn (IP Hidden, New member, 1)
Date: June 27, 2005 04:28AM
Hi,
I am performing the immunoprecipitaion but I am meeting a problem. Could you please give me some solutions? My experiment was performed as following: 5 ug of the extract (containing antigen) + 1ug of primary antibody + 50 mM Tris, pH7.5, total volume of reaction was 300 ul, mixture was incubated at 4oC overning.Then add 50 ul of Protein G magnetic beads to the reaction mixture. This mixture was incubated continuously for 1 hour at 4oC. Mixture was applied onto the column (using MAS kit). Most of the interested protein was appeared in the Flow through. Nothing is in the wash, small amount of interested protein is in the eluent. That mean antigen could not bind to the primary antibody. Could you please give me some solutions to solve this problem? Thank you very much!
Re: Immunoprecipitation Troubleshooting
Posted by: Jonathan W (IP Hidden, Unregistered user, )
Date: June 27, 2005 08:01AM
hi,
It seems to me the amount of protein is too low (5ug?) to do an immunoprecipitation. However I have no experience using the magnetic beads but you can check the recommended protein quantity. Antibody is also very critical in performing IP. You should chose an antibody that is suitable for IP. Check your Ab to see if it can be applied on IP since some antibody only recognize the hidden epitopes which are exposed only after denaturing. Good luck.
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